1. Field of the Invention
The present invention relates to a novel method for producing formulations comprising a polynucleotide, a block copolymer and cationic surfactant. The formulations produced by the current method are suitable for use in polynucleotide based medicaments.
2. Related Art
The use of non-ionic block copolymers as adjuvants in gene and vaccine delivery has been documented in the art. Newman et al. (Critical Reviews in Therapeutic Drug Carrier Systems 15 (2): 89-142 (1998)) review a class of non-ionic block copolymers which show adjuvant activity. The basic structure comprises blocks of polyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-POP-POE block copolymer. Newman et al. id., disclose that certain POE-POP-POE block copolymers may be useful as adjuvants to an influenza protein-based vaccine, namely higher molecular weight POE-POP-POE block copolymers containing a central POP block having a molecular weight of over about 9000 daltons to about 20,000 daltons and flanking POE blocks which comprise up to about 20% of the total molecular weight of the copolymer (See U.S. Reissue Pat. No. 36,665, U.S. Pat. No. 5,567,859, U.S. Pat. No. 5,691,387, U.S. Pat. No. 5,696,298 and U.S. Pat. No. 5,990,241, all issued to Emanuele, et al., regarding these POE-POP-POE block copolymers). Published International Patent Application No. WO 96/04932 further discloses higher molecular weight POE/POP block copolymers which have surfactant characteristics and show biological efficacy as vaccine adjuvants.
U.S. Pat. No. 5,656,611 and Published International Patent Application No. WO 99/06055 disclose compositions which include a polynucleotide, and a block copolymer containing a non-ionic portion and a polycationic portion. A surfactant is added to increase solubility and the end result is the formation of micelles. This formulation allows stabilization of polynucleic acids and enhances transfection efficiency. Published International Patent Application No. WO 99/21591 discloses a soluble ionic complex comprising an aqueous mixture of a polynucleotide and a benzylammonium group-containing cationic surfactant and the use of this complex in vaccine and gene delivery.
Recent disclosure in Published International Patent Application No. WO 02/00844, hereby incorporated in its entirety by reference, describes polynucleotide vaccine adjuvants which comprise a polynucleotide, a block copolymer and a cationic surfactant. By including the cationic surfactant in the formulation, the percentage of polynucleotide that is associated with the block copolymer/cationic surfactant adjuvant is increased. In addition, this formulation has demonstrated enhanced in vivo immune response to polynucleotide vaccines and/or gene therapy-based transgenes.
However, the method described in Published International Patent Application No. WO 02/00844 to produce this polynucleotide/block copolymer/cationic surfactant composition requires thermally cycling the mixture several times through the cloud point of the block copolymer to form the polynucleotide complexes. These multiple heating and cooling cycles are expensive and time consuming, especially when considering the production of large quantities of the formulation required during commercial manufacturing. In addition, no sterilization step was disclosed in WO 02/00844. The requirement to sterilize all components prior to mixing and producing the formulation under sterile conditions increases the cost of large-scale production considerably and hinders the ability to scale up the production of this formulation for commercial manufacturing.
Therefore, a need remains in the art for a method of producing sterile formulations as described above, that also allow for a scalable production platform.